PROJECT SUMMARY/ABSTRACT Numerous life forms including bacteria, fungi, plants, and animals involuntarily regulate their metabolism, physiology, and behavior in anticipation of sunrise and sunset. These circadian rhythms arise from endogenous molecular timekeeping systems, called circadian clocks, which generate waves of assembly and disassembly of clock components. Unknown are the temporal and causal relationships between these transitory events, and how they are important to clock function. The objective of the LiWang lab is to elucidate the dynamic mechanism by which these waves are produced by and transmitted from the circadian oscillator, through clock-output pathways, to transcription factor-DNA binding rhythms. The approach is to carry out real-time measurements in vitro on the cyanobacterial circadian clock, reconstituted in its entirety (six proteins and DNA) outside the milieu of live cells. Despite the availability of a near-complete set of high-resolution structures of cyanobacterial clock protein complexes (manuscript under review at Science, submitted on 06/04/16), the dynamic mechanism of this system remains obscure. Thus, the LiWang lab is conducting real-time experiments on reconstituted cyanobacterial circadian clock reactions to unravel the dynamic timekeeping mechanism of this system. In aim 1, the LiWang lab will elucidate the temporal and causal relationships between transitory interaction events at the molecular level, using real-time nuclear magnetic resonance spectroscopy (NMR). Time signals will be followed as they propagate from the KaiABC oscillator, through the antagonistic output pathways, mediated by SasA and CikA proteins, to generate circadian rhythms of RpaA-DNA binding. In aim 2, the temporally regulated signal transmission network involving specific residues across KaiC (the heart of the clock) will be mapped, from sites of phosphorylation to the KaiA-binding sites (daytime) and KaiB- and SasA-binding sites (nighttime), using real-time NMR. Preliminary data on both aims demonstrate feasibility. This proposal is innovative, because for the first time, it will be possible to observe the rippling of time signals across the lengths of circadian clock proteins and their transmission from one clock component to the next, cycle after circadian cycle, in real time. This proposal is significant, because resolving temporal interactions within and between clock components will reveal the dynamic machinery that generates and propagates waves of clock signals, and thereby provide insights into biological timekeeping that are unobtainable from static structures.